Preparation Of Cucurbitacin B Solid Dispersion And Its Pharmacokinetic In Rats

Cucurbitacins is a rich content of highly oxidized tetracyclic triterpenoid isolated from Cucurbitaceae,the main component of which is cucurbitacin B.The cucurbitacin B has many obvious disadvantages,such as insoluble in water,poor absorption in the stomach and intestines and low bioavailability.In order to improve the dissolution of cucurbitacin B and increase the bioavailability,the solid dispersion technology was used to prepare solid dispersions by using the solvent Losham method as the auxiliary material of poloxamer 407.The SEM,DSC,X-RD,FTIR and other means were used to characterize the dissolution.The dissolution test was used to compare the water solubility of the solid dispersion,the physical mixture and the drug substance.Rats were used as experimental animals to compare the pharmacokinetics of solid dispersions of cucurbitacin B and drug substances,and to explore the differences between the main pharmacokinetic parameters.Objective:A method for the dissolution test of cucurbitacin B in vitro was established.UPLC-MS/MS method was used to determine the cucurbitacin B content in rat plasma.The solid dispersion was prepared by the solvent method and its physicochemical properties were characterized.The pharmacokinetics and bioavailability of the solid dispersion of cucurbitacin B were studied.Methods:1.A UV method was used to determine the content of cucurbitacin B and its content in solid dispersion.The dissolution rate of cucurbitacin B solid dispersion at different time was determined by HPLC and its methodological investigation was carried out.The dissolution test conditions of the solid dispersion of cucurbitacin B were screened by examining the conditions of the dissolution medium,rotation speed,and the like.2.Establishment of an analytical method for the determination of cucurbitacin B in rat plasma by UPLC-MS/MS method:optimization of mass spectrometry conditions,screening and optimization of biological sample pretreatment methods;methodology review,linear range,precision and accuracy,recovery Rate,matrix effect and stability.3.Determination of Absolute Bioavailability of cucurbitacin B:cucurbitacin B was given to rats by injecting and gavage respectively.UPLC-MS/MS method was used to determine the cucurbitacin B content in rat plasma at different time points.The main pharmacokinetic parameters were calculated using DAS 3.2.8 software and the absolute bioavailability of cucurbitacin B was calculated.4.The solid dispersion carrier was screened by the crystal inhibition test and the solubility test;the solid dispersion was prepared by the solvent method,and the drug loading ratio and solvent were screened by the single factor method.5.Scanning electron microscopy,differential scanning calorimetry,powder X-ray diffractometry,and Fourier infrared spectroscopy were used to identify the cucurbitacin B solid dispersion.A dissolution test was used to evaluate the dissolution of the solid dispersion of cucurbitacin B.6.Study of cucurbitacin B solid dispersions of pharmacokinetics in rats:Taking rats as experimental animals,cucurbitacin B raw material and solid dispersion were given by the single intragastric administration.UPLC-MS/MS method was used to determine the concentration of cucurbitacin B in plasma and to draw the plasma concentration-time curve,in which plasma was taken from the eyelid at different time points.DAS 3.2.8 was used to fit the major pharmacokinetic parameters,to calculate relative bioavailability,and to compare the differences in pharmacokinetic parameters of solid dispersion and cucurbitacin B.Results:1.An UV method was used to determine the content of cucurbitacin B,which the detection wavelength was 228 nm.the linear range of cucurbitacin B was 3.84³2.64?g/mL,the linear regression equation was A=0.0255C+0.0799 and the correlation coefficient was 0.999.The precision RSD was 3.59%,the average recoveries of samples with low,medium and high concentrations were 99.38%,99.21%and 100.20%,respectively.The sample was stable within 24 h.The dissolution rate was determined by HPLC method.The linear range of cucurbitacin B was 0.27³.78?g/mL,the linear regression equation was Y=24070.4X+710.9,and the correlation coefficient was 0.9999.The RSDs of the low,medium,and high concentrations of the three samples were 3.78%,3.19%,and 2.66%,respectively.The average recoveries of the low,medium,and high dissolved samples were 97.57%,100.64%,and 99.26%,respectively.The sample was stable within 24 h.The dissolution conditions of cucurbitacin B solid dispersion were as follows:pure water was used as dissolving medium,paddle method,rotation speed was 100r/min,and the temperature was 37?.2.UPLC-MS/MS method was used to determine the content of cucurbitacin B in plasma.The linear range of cucurbitacin B in plasma was0.05¹000 ng/mL,the linear regression equation was Y=15801.4X+3365.1,and the correlation coefficient was 0.9996,and the minimum limit of quantitation was 0.05 ng/mL.The average accuracy of the LLOQ sample was110.67%and the precision RSD was 5.32%.The average accuracies of the QC samples were 98.17%,104.50%,and 99.67%respectively.The precision RSDs were 4.66%,5.44%,and 5.52%,respectively.The matrix effect of B was 94.19⁹9.42%,the matrix effect of IS was 100.83%,the recovery rate was more than 85%,and the stability of the sample was good.3.The absolute bioavailability of cucurbitacin B was 1.37%.4.The results of suppression test showed that Poloxamer 407 is significantly superior to F68,PEG-6000,Soluplus,HPMC E₃₀,and PVP K₉₀ in the ability to inhibit crystallization of cucurbitacin B and the solubility of the cucurbitacin B bulk drug in the poloxamer 407 solution is increased by about9%compared to the solubility in the pure water.Poloxamer 407 was selected as the carrier,and the solid dispersion of cucurbitacin B was prepared by the solvent method according to the loading ratio of 1:5 and 1:7 with dichloromethane as the solvent.When dissolved for 45 min,the dissolution rates of API,physical mixture,1:5 solid dispersion and 1:7 solid dispersion were 12.92%,30.61%,88.58%,98.34%,respectively.5.Cucurbitacin B solid dispersion was white powder with no agglomeration and no blocking.Scanning electron microscopy shows that the raw materials of cucurbitacin B are prismatic crystals,poloxamer 407 is in an irregular mass.There are columnar crystals and irregular lumps in the physical mixture.However,the solid dispersion is obviously different from the above structure in which crystal structure of cucurbitacin B is not found.The results of X-RD detection manifested that there were obvious crystal diffraction peaks at 7.0°,13.5°,16.1°,20.5°for the cucurbitacin B bulk drug,and the characteristic diffraction peak of cucurbitacin B disappeared in the solid dispersion.DSC analysis showed that endothermic peaks appeared at181.75?and 61.31?for cucurbitacin B and poloxamer 407,respectively.The above two characteristics endothermic peaks appeared in the physical mixture,characteristics of poloxamer 407 endothermic peaks appeared only in solid dispersion.The FTIR results are as follows:The characteristic infrared spectra of cucurbitacin B are characteristic peak of ketone(1720 cm^-1,1695cm^-1),and hydroxy characteristic peak(3504 cm^-1).Based on the above analysis,it is presumed that the drug in the solid dispersion exists in an amorphous state with microcrystals.6.The relative bioavailability of solid dispersion in the proportion of 1:5and 1:7 was 268.35%and 364.02%,respectively.The T_max of cucurbitacin B solid dispersion administered by gavage in rats was 1h earlier than that of the drug.The AUC of the solid dispersion group of 1:5 and 1:7 was significantly higher than that of the API Group?P<0.05?.The C_max in the 1:5 and 1:7proportion of the solid dispersion group was significantly higher than that in the API Group?P<0.05?.Both CL_z/F and V_z/F were lower than those of the drug substance group.Conclusion:The solid dispersion of cucurbitacin B prepared by the solvent method can significantly increase the dissolution rate.The pharmacokinetics study showed that solid dispersion can significantly improve its bioavailability.